Enhanced pyruvate to acetolactate conversion in yeast

ABSTRACT

A high flux in conversion of pyruvate to acetolactate was achieved in yeast through expression of acetolactate synthase in the cytosol in conjunction with reduction in pyruvate decarboxylase activity. Additional manipulations to improve flux to acetolactate are reduced pyruvate dehydrogenase activity and reduced glycerol-3-phosphate dehydrogenase activity. Production of compounds having acetolactate as an upstream intermediate benefit from the increased conversion of pruvate to acetolactate in the described strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/299,954, filed Nov. 18, 2011, now U.S. Pat. No. 8,669,094 which is a continuation of U.S. application Ser. No. 12/477,942, filed Jun. 4, 2009, which claims the benefit of U.S. Provisional Application No. 61/058,970, filed Jun. 5, 2008, each of which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name: CL4209USCNT2sequencelisting.txt; Size: 596,674 bytes; and Date of Creation: Jan. 16, 2014) is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology and the metabolism of yeast. More specifically, engineering yeast for a high flux through an acetolactate intermediate allows increased production of compounds in pathways including acetolactate as an upstream substrate.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase. 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). 2,3-butanediol may be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol may be used as plasticizers (Voloch et al. Fermentation Derived 2,3-Butanediol, in Comprehensive Biotechnology, Pergamon Press Ltd, England Vol 2, Section 3:933-947 (1986)).

Microorganisms may be engineered for expression of biosynthetic pathways for production of 2,3-butanediol, 2-butanone, 2-butanol and isobutanol. Commonly owned and co-pending US Patent Application publication US 20070092957 A1 discloses the engineering of recombinant microorganisms for production of isobutanol. Commonly owned and co-pending US Patent Application publications US 20070259410A1 and US 20070292927A1 disclose the engineering of recombinant microorganisms for production of 2-butanone or 2-butanol. Multiple pathways are disclosed for biosynthesis of isobutanol and 2-butanol, all of which initiate with cellular pyruvate. Butanediol is an intermediate in the 2-butanol pathway disclosed in commonly owned and co-pending US Patent Application publication US 20070292927A1.

Production of 2,3-butanediol, 2-butanone, 2-butanol and isobutanol in recombinant yeasts is limited by availability of substrate flow from natural yeast metabolic pathways into engineered biosynthetic pathways producing these compounds. Since the biosynthetic pathways for isobutanol, 2,3-butanediol, 2-butanol, and 2-butanone draw from host cell production of pyruvate, this substrate may be a limitation in product formation. The first step in these engineered pathways is conversion of pyruvate to acetolactate, which is catalyzed by acetolactate synthase.

Pyruvate metabolism has been altered in yeast for production of lactic acid and glycerol. US20070031950 discloses a yeast strain with a disruption of one or more pyruvate decarboxylase or pyruvate dehydrogenase genes and expression of a D-lactate dehydrogenase gene, which is used for production of D-lactic acid. Ishida et al. (Biosci. Biotech. and Biochem. 70:1148-1153 (2006)) describe Saccharomyces cerevisiae with disrupted pyruvate dehydrogenase genes and expression of lactate dehydrogenase. US2005/0059136 discloses glucose tolerant C₂ carbon source-independent (GCSI) yeast strains with no pyruvate decarboxylase activity, which may have an exogenous lactate dehydrogenase gene. Nevoigt and Stahl (Yeast 12:1331-1337 (1996) describe the impact of reduced pyruvate decarboxylase and increased NAD-dependent glycerol-3-phosphate dehydrogenase in Saccharomyces cerevisiae on glycerol yield.

To improve the production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone in yeast, the problem remaining to be solved is to increase the conversion of pyruvate to acetolactate, which then flows into engineered biosynthetic pathways to produce these compounds.

SUMMARY OF THE INVENTION

The invention describers the finding that that by combining expression of acetolactate synthase enzyme activity in the yeast cytosol with reduced pyruvate decarboxylase activity, a surprisingly high flux from pyruvate to acetolactate can be achieved. The invention provides yeast cells that are engineered to have high conversion of endogenous pyruvate to acetolactate in the cytoplasm due to suppression of competing metabolic pathways in the presence of cytosolic acetolactate synthase activity. The yeast cells may also have an engineered complete biosynthetic pathway for production of isobutanol, 2,3-butanediol, 2-butanone or 2-butanol. The engineered yeast may be used for production of isobutanol, 2,3-butanediol, 2-butanone or 2-butanol, or other products derived from acetolactate such as valine, isoleucine and isoamyl alcohol.

Accordingly the invention provides a recombinant yeast cell comprising at least one gene encoding a cytosol-localized polypeptide having acetolactate synthase activity wherein the yeast cell is substantially free of an enzyme having pyruvate decarboxylase activity, and wherein the cell converts pyruvate to acetolactate. Preferred recombinant yeast cells of the invention are those having disruptions in genes encoding pyruvate decarboxylases, pyruvate dehydrogenases and NAD-dependent glycerol-3-phosphate dehydrogenases.

In other embodiments the invention provides recombinant yeast cells having the ability to produce 2,3-butanediol, isobutanol, 2-butanone or 2-butanol comprising at least one gene encoding a cytosol-localized polypeptide having acetolactate synthase activity wherein the yeast cell is substantially free of an enzyme having pyruvate decarboxylase activity, and wherein the cell converts pyruvate to acetolactate with at least about 60% of theoretical yield.

In another embodiment the invention provides methods for the production of 2,3-butanediol, isobutanol, 2-butanone or 2-butanol comprising growing the recombinant yeast cells of the invention under conditions wherein 2,3-butanediol, isobutanol, 2-butanone or 2-butanol is produced and optionally recovering the 2,3-butanediol, isobutanol, 2-butanone or 2-butanol.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understood from the following detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.

FIG. 1 shows pathways and enzymes for pyruvate utilization.

FIG. 2 shows three different isobutanol biosynthetic pathways.

FIG. 3 shows four different 2-butanol biosynthetic pathways.

FIGS. 4A-4D show sequence relationships of acetolactate synthase (als) coding regions that were retrieved by BLAST analysis using the sequence of B. subtilis AlsS, limiting to the 100 closest neighbors. The als encoding sequence is identified by its source organism.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

TABLE 1 SEQ ID Numbers of Expression Coding Regions and Proteins SEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid Klebsiella pneumoniae budB (acetolactate 1  2 synthase) Bacillus subtilis alsS 3  4 (acetolactate synthase) Lactococcus lactis als 5  6 (acetolactate synthase) Als Staphylococcus aureus 7  8 Als Listeria monocytogenes 9 10 Als Streptococcus mutans 11 12 Als Streptococcus thermophilus 13 14 Als Vibrio angustum 15 16 Als Bacillus cereus 17 18 budA, acetolactate decarboxylase from 19 20 Klebsiella pneumoniae ATCC 25955 alsD, acetolactate decarboxylase from 21 22 Bacillus subtilis budA, acetolactate decarboxylase from 23 24 Klebsiella terrigena budC, butanediol dehydrogenase from 25 26 Klebsiella pneumoniae IAM1063 butanediol dehydrogenase from 27 28 Bacillus cereus butB, butanediol dehydrogenase from 29 30 Lactococcus lactis RdhtA, B12-indep diol dehydratase from 31 32 Roseburia inulinivorans RdhtB, B12-indep diol dehydratase 33 34 reactivase from Roseburia inulinivorans sadB, butanol dehydrogenase from 35 36 Achromobacter xylosoxidans S. cerevisiae ILV5 37 38 (acetohydroxy acid reductoisomerase) Vibrio cholerae ketol-acid 39 40 reductoisomerase Pseudomonas aeruginosa ketol-acid 41 42 reductoisomerase Pseudomonas fluorescens ketol-acid 43 44 reductoisomerase S. cerevisiae ILV3 45 46 (Dihydroxyacid dehydratase; DHAD) Lactococcus lactis kivD (branched-chain α- 47 48 keto acid decarboxylase), codon optimized Lactococcus lactis kivD (branched-chain α- 49  48* keto acid decarboxylase) Pf5.llvC-Z4B8 mutant Pseudomonas 168 169  fluorescens acetohydroxy acid reductoisomerase Bacillis subtilis kivD codon optimized for 172 173  S. cerevisiae expression Equus caballus alcohol dehydrogenase 174 175  codon optimized for S. cerevisiae expression Streptococcus mutans ilvD (DHAD) 185 186  *The same amino acid sequence is encoded by SEQ ID NOs: 47 and 49.

TABLE 2 SEQ ID Numbers of Disruption target Gene coding regions and Proteins SEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid PDC1 pyruvate decarboxylase from 50 51 Saccharomyces cerevisiae PDC5 pyruvate decarboxylase from 52 53 Saccharomyces cerevisiae PDC6 pyruvate decarboxylase from 54 55 Saccharomyces cerevisiae pyruvate decarboxylase from 56 57 Candida glabrata PDC1 pyruvate decarboxylase from 58 59 Pichia stipitis PDC2 pyruvate decarboxylase from 60 61 Pichia stipitis pyruvate decarboxylase from 62 63 Kluyveromyces lactis pyruvate decarboxylase from 64 65 Yarrowia lipolytica pyruvate decarboxylase from 66 67 Schizosaccharomyces pombe GPD1 NAD-dependent glycerol-3- 68 69 phosphate dehydrogenase from Saccharomyces cerevisiae GPD2 NAD-dependent glycerol-3- 70 71 phosphate dehydrogenase from Saccharomyces cerevisiae GPD1 NAD-dependent glycerol-3- 72 73 phosphate dehydrogenase from Pichia stipitis GPD2 NAD-dependent glycerol-3- 74 75 phosphate dehydrogenase from Pichia stipitis NAD-dependent glycerol-3-phosphate 76 77 dehydrogenase from Kluyveromyces thermotolerans GPD1 NAD-dependent glycerol-3- 78 79 phosphate dehydrogenase from Schizosaccharomyces pombe GPD2 NAD-dependent glycerol-3- 80 81 phosphate dehydrogenase from Schizosaccharomyces pombe PDA1, Pyruvate dehydrogenase from 82 83 Saccharomyces cerevisiae PDB1, Pyruvate dehydrogenase from 84 85 Saccharomyces cerevisiae Lat1 pyruvate dehydrogenase complex 86 87 from Saccharomyces cerevisiae Lpd1 pyruvate dehydrogenase complex 88 89 from Saccharomyces cerevisiae Pdx1 pyruvate dehydrogenase complex 90 91 from Saccharomyces cerevisiae PDA1, Pyruvate dehydrogenase from 92 93 Pichia stipitis PDB1, Pyruvate dehydrogenase from 94 95 Pichia stipitis Pyruvate dehydrogenase from 96 97 Kluyveromyces lactis PDA1, Pyruvate dehydrogenase from 98 99 Schizosacharomyces pombe PDB1, Pyruvate dehydrogenase from 100 101 Schizosacharomyces pombe

SEQ ID NOs:102-113, 117-134, 127-134, 136, 137, 139-164, 178, 179, 181, 182, 189-197, 199-205 and 207-208 are sequencing and PCR primers used and described in the Examples.

SEQ ID NO:114 is the S. cerevisiae GPD1 promoter.

SEQ ID NO:115 is the S. cerevisiae CYC1 terminator.

SEQ ID NO:116 is the S. cerevisiae FBA promoter.

SEQ ID NO:125 is the S. cerevisiae CUP1 promoter.

SEQ ID NO:126 is the S. cerevisiae ADH1 terminator.

SEQ ID NO:135 is the S. cerevisiae GPM1 promoter.

SEQ ID NO:138 is the sequence of a synthetic fragment containing coding regions for Roseburia inulinivorans B₁₂-independent diol dehydratase and reactivase.

SEQ ID NO:143 is the dual terminator.

SEQ ID NO:165 is the sequence of the pLH475-Z4B8 vector.

SEQ ID NO:166 is the S. cerevisiae CYC1-2 terminator.

SEQ ID NO:167 is the S. cerevisiae ILV5 promoter.

SEQ ID NO:170 is the S. cerevisiae ILV5 terminator.

SEQ ID NO:171 is the sequence of the pLH468 vector.

SEQ ID NO:176 is the sequence of the pNY8 vector.

SEQ ID NO:177 is the S. cerevisiae GPD1-2 promoter.

SEQ ID NO:180 is the sequence of the pRS425::GPM-sadB vector.

SEQ ID NO:183 is the sequence of the pRS423 FBA ilvD(Strep) vector.

SEQ ID NO:184 is the S. cerevisiae FBA terminator.

SEQ ID NO:187 is the sequence of the GPM-sadB-ADHt fragment.

SEQ ID NO:188 is the sequence of the pUC19-URA3r vector.

SEQ ID NO: 198 is the sequence of the ilvD-FBA1t fragment.

SEQ ID NO:206 is the sequence of the URA3r2 marker template DNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to recombinant yeast cells engineered for improved production of acetolactate and compounds having acetolactate as an upstream intermediate including isobutanol, 2,3-butanediol, 2-butanone and 2-butanol. In addition, the present invention relates to methods of producing these compounds using the present engineered yeast cells. Isobutanol, 2,3-butanediol, 2-butanone and 2-butanol are important compounds for use in replacing fossil fuels either directly or as intermediates for further chemical synthesis, in addition to applications as solvents and/or extractants.

The following abbreviations and definitions will be used for the interpretation of the specification and the claims.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “butanol” as used herein, refers to 2-butanol, 1-butanol, isobutanol, or mixtures thereof.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to an enzyme pathway to produce 2-butanone from pyruvate.

The terms “acetolactate synthase” and “acetolactate synthetase” are used interchangeably herein to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO₂. Preferred acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (DNA: SEQ ID NO:3; protein: SEQ ID NO:4), Klebsiella pneumoniae (DNA: SEQ ID NO:1; protein: SEQ ID NO:2), and Lactococcus lactis (DNA: SEQ ID NO:5; protein: SEQ ID NO:6).

The term “acetolactate decarboxylase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (DNA: SEQ ID NO:21, Protein: SEQ ID NO:22), Klebsiella terrigena (DNA: SEQ ID NO:23, Protein: SEQ ID NO:24) and Klebsiella pneumoniae (DNA: SEQ ID NO:19, protein: SEQ ID NO:20).

The term “butanediol dehydrogenase” also known as “acetoin reductase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (DNA: SEQ ID NO:25, protein: SEQ ID NO:26). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (DNA: SEQ ID NO:27, protein: SEQ ID NO:28), and Lactococcus lactis (DNA: SEQ ID NO:29, protein: SEQ ID NO:30).

The term “substantially free” when used in reference to the presence or absence of enzyme activities (pyruvate decarboxylase, pyruvate dehydrogenase, NAD-dependent glycerol-3-phosphate dehydrogenase) in carbon pathways that compete with the present isobutanol pathway means that the level of the enzyme is substantially less than that of the same enzyme in the wildtype host, where less than about 20% of the wildtype level is preferred and less than about 10% of the wildtype level is more preferred. The activity may be less than about 5% or 1% of wildtype activity.

The term “a facultative anaerobe” refers to a microorganism that can grow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides. The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” or “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA). Expression may also refer to translation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of a nucleic acid molecule into a host cell, which may be maintained as a plasmid or integrated into the genome. Host cells containing the transformed nucleic acid molecules are referred to as “transgenic” or “recombinant” or “transformed” cells.

The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon degeneracy.”

The term “codon-optimized” as it refers to coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. “Codon-bias” can be exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a coding region for improved expression in a host cell, it is desirable to design the coding region such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

As used herein, an “isolated nucleic acid fragment” or “isolated nucleic acid molecule” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 24% to 100% may be useful in describing the present invention, such as 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).

Production Through Acetolactate from Endogenous Pyruvate

Yeast cells produce pyruvate from sugars, which is then utilized in a number of pathways of cellular metabolism including those shown in FIG. 1. Yeast cells can be engineered to produce a number of desirable products with the initial biosynthetic pathway step being conversion of endogenous pyruvate to acetolactate. Engineered biosynthetic pathways for synthesis of isobutanol (see FIG. 2) are described in commonly owned and co-pending US Patent Application Publication US20070092957, which is herein incorporated by reference, and for synthesis of 2-butanol and 2-butanone (see FIG. 3) are described in commonly owned and co-pending US Patent Application Publications US20070259410 and US 20070292927, which are herein incorporated by reference. The product 2,3-butanediol is an intermediate in the biosynthetic pathway described in US 20070292927. Each of these pathways has the initial step of converting pyruvate to acetolactate by acetolactate synthase. Therefore product yield from these biosynthetic pathways will in part depend upon the amount of acetolactate that can be produced from pyruvate and the amount of pyruvate that is available.

Applicants have discovered that by combining expression of acetolactate synthase enzyme activity in the yeast cytosol with reduced pyruvate decarboxylase activity, a surprisingly high flux from pyruvate to acetolactate can be achieved. Flux from pyruvate to acetolactate may be measured by conversion of glucose or sucrose to 2,3-butanediol. Pyruvate is produced from glucose or sucrose. Synthesis of 2,3-butanediol requires two additional steps: conversion of acetolactate to acetoin by acetolactate decarboxylase, and conversion of acetoin to 2,3-butanediol by butanediol dehydrogenase. Thus at least as much flux from pyruvate to acetolactate must occur as the measured flux from glucose or sucrose to 2,3-butanediol, and potentially more since the two enzymatic steps following acetolactate are likely to be less than 100% efficient.

Applicants found that about 86% of the theoretical yield of sucrose conversion to 2,3-butanediol was achieved in the presence of an electron sink, as shown in Example 4 herein. About 90% of the theoretical yield of glucose conversion to 2,3-butanediol was achieved in the presence of an electron sink. The theoretical yield of glucose to 2,3-butanediol is calculated to be 0.5 g of 2,3-butanediol per 1 g of glucose. An electron sink is required for redox balance in the biosynthetic pathway to 2,3-butanediol. Complete 2-butanol and isobutanol biosynthetic pathways that are disclosed in US Patent Publications US20070092957, US20070259410, and US 20070292927, are in themselves redox balanced and require no additional electron sink to reach maximal product formation.

Glucose conversion to 2,3-butanediol without an added electron sink was found herein to be about 60% of theoretical yield (0.3 g of 2,3-butanediol per 1 g of glucose), and conversion was about 68% from sucrose. Thus the conversion of pyruvate to acetolactate achieved was at least about 60%. Therefore by combining expression of acetolactate synthase enzyme activity in the yeast cytosol with reducing pyruvate decarboxylase activity, without balance of redox equivalents, conversion of pyruvate to acetolactate may achieve at least about 60% of theoretical yield. By combining expression of acetolactate synthase enzyme activity in the yeast cytosol with reducing pyruvate decarboxylase activity, and with balance of redox equivalents, conversion of pyruvate to acetolactate may achieve at least about 86% of theoretical yield.

Expression of Acetolactate Synthase in the Yeast Cytosol

Endogenous acetolactate synthase in yeast is encoded in the mitochondrial genome and expressed in the mitochondria. To prepare a recombinant yeast strain of the present invention a genetic modification is made that provides cytosolic expression of acetolactate synthase. Acetolactate synthase is expressed from the nucleus and no mitochondrial targeting signal is included so that the enzyme remains in the cytosol (cytosol-localized).

Acetolactate synthase enzymes, which also may be called acetohydroxy acid synthase, belong to EC 2.2.1.6 (switched from 4.1.3.18 in 2002), are well-known, and they participate in the biosynthetic pathway for the proteinogenic amino acids leucine and valine, as well as in the pathway for fermentative production of 2,3-butanediol and acetoin in a number of organisms.

The skilled person will appreciate that polypeptides having acetolactate synthase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some examples of suitable acetolactate synthase enzymes are available from a number of sources, as described in the definitions above. Acetolactate synthase enzyme activities that have substrate preference for pyruvate over ketobutyrate are of particular utility, such as those encoded by alsS of Bacillus and budB of Klebsiella (Gollop et al., J. Bacteriol. 172(6):3444-3449 (1990); Holtzclaw et al., J. Bacteriol. 121(3):917-922 (1975)).

Because acetolactate synthases are well known, and because of the prevalence of genomic sequencing, suitable acetolactate synthases may be readily identified by one skilled in the art on the basis of sequence similarity using bioinformatics approaches. Typically BLAST (described above) searching of publicly available databases with known acetolactate synthase amino acid sequences, such as those provided herein, is used to identify acetolactate synthases, and their encoding sequences, that may be used in the present strains. For example, acetolactate synthases that are the 100 closest neighbors of the B. subtilis AlsS sequence are depicted in a phylogenetic tree in FIGS. 4A-4D. The homology relationships between the sequences identified are shown in this tree. Among these sequences are those having 40% identity, yet these have been verified as acetolactate synthases. A representative sequence from each bracket is given in Table 2. Acetolactate synthase proteins having at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to any of the acetolactate synthase proteins in Table 1 (SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18), or any of the acetolactate synthase proteins represented in FIGS. 4A-4D may be used in the present strains. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Examples of sequences encoding acetolactate synthase which may be used to provide cytosolic expression of acetolactate synthase activity are listed in Table 1 (SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17). Additional acetolactate synthase encoding sequences that may be used for yeast cytosolic expression may be identified in the literature and in bioinformatics databases well known to the skilled person, and some coding regions for als proteins are represented in FIGS. 4A-4D by the source organism. Any acetolactate synthase having EC number 2.2.1.6 may be identified by one skilled in the art and may be used in the present strains.

Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature. For example each of the acetolactate synthase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

For example, genes encoding similar proteins or polypeptides to the acetolactate synthase encoding genes described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.

Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

Alternatively, the described acetolactate synthase encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCI, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.

Cytosolic expression of acetolactate synthase is achieved by transforming with a gene comprising a sequence encoding an acetolactate synthase protein, with no mitochondrial targeting signal sequence. Methods for gene expression in yeasts are known in the art (see for example Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding an acetolactate synthase, including, but not limited to constitutive promoters FBA, GPD1, ADH1, and GPM, and the inducible promoters GAL1, GAL10, and CUP1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1.

Suitable promoters, transcriptional terminators, and acetolactate synthase coding regions may be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells as described in Examples 2-4. These vectors allow strain propagation in both E. coli and yeast strains. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2μ origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Construction of expression vectors with a chimeric gene encoding an acetolactate synthase may be performed by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast. Typically, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain a ≧21 bp sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X’, a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g. TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. Typically, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding regionX-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.

Reduced Pyruvate Decarboxylase Activity

Endogenous pyruvate decarboxylase activity in yeast converts pyruvate to acetaldehyde, which is then converted to ethanol or to acetyl-CoA via acetate (see FIG. 1). Yeasts may have one or more genes encoding pyruvate decarboxylase. For example, there is one gene encoding pyruvate decarboxylase in Kluyveromyces lactis, while there are three isozymes of pyruvate decarboxylase encoded by the PDC1, PCD5, and PDC6 genes in Saccharomyces cerevisiae, as well as a pyruvate decarboxylase regulatory gene PDC2. Expression of pyruvate decarboxylase from PDC6 is minimal. In the present yeast strains the pyruvate decarboxylase activity is reduced by disrupting at least one gene encoding a pyruvate decarboxylase, or a gene regulating pyruvate decarboxylase gene expression. For example, in S. cerevisiae the PDC1 and PDC5 genes, or all three genes, are disrupted. In addition, pyruvate decarboxylase activity may be reduced by disrupting the PDC2 regulatory gene in S. cerevisiae. In other yeasts, genes encoding pyruvate decarboxylase proteins such as those having at least about 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to PDC1 or PDC5 may be disrupted.

Examples of yeast strains with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported such as for Saccharomyces in Flikweert et al. (Yeast (1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol. (1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann, (Mol Gen Genet. (1993) 241:657-666). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC with Accession #200027 and #200028.

Expression of pyruvate decarboxylase genes may be reduced in any yeast strain that is also engineered with cytosolic acetolactate synthase expression and other biosynthetic pathway enzyme encoding genes for production of a compound derived from acetolactate. Examples of yeast pyruvate decarboxylase genes that may be targeted for disruption are listed in Table 2 (SEQ ID NOs:50, 52, 54, 56, 58, 60, 62, 64, 66). Other target genes, such as those encoding pyruvate decarboxylase proteins having at least about 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to the pyruvate decarboxylases listed in Table 2 (SEQ ID NOs:51, 53, 55, 57, 59, 61, 63, 65, 67) may be identified in the literature and in bioinformatics databases well known to the skilled person. Additionally, the sequences described herein or those recited in the art may be used to identify homologs in other yeast strains, as described above for identification of acetolactate synthase encoding genes.

Alternatively, because pyruvate decarboxylase encoding sequences are well known, and because sequencing of the genomes of yeasts is prevalent, suitable pyruvate decarboxylase gene targets may be identified on the basis of sequence similarity using bioinformatics approaches. Genomes have been completely sequenced and annotated and are publicly available for the following yeast strains: Ashbya gossypii ATCC 10895, Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c, Schizosaccharomyces pombe 972h-, and Yarrowia lipolytica CLIB122. Typically BLAST (described above) searching of publicly available databases with known pyruvate decarboxylase encoding sequences or pyruvate decarboxylase amino acid sequences, such as those provided herein, is used to identify pyruvate decarboxylase encoding sequences of other yeasts.

Accordingly it is within the scope of the invention to provide pyruvate decarboxylase proteins having at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to any of the pyruvate decarboxylase proteins disclosed herein (SEQ ID NO:51, 53, 55, 57, 59, 61, 63, 65, and 67) Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Genes encoding pyruvate decarboxylase may be disrupted in any yeast cell using genetic modification. Many methods for genetic modification of target genes are known to one skilled in the art and may be used to create the present yeast strains. Modifications that may be used to reduce or eliminate expression of a target protein are disruptions that include, but are not limited to, deletion of the entire gene or a portion of the gene encoding a pyruvate decarboxylase, inserting a DNA fragment into a pyruvate decarboxylase encoding gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into a pyruvate decarboxylase coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into a pyruvate decarboxylase coding region to alter amino acids so that a non-functional or a less enzymatically active protein is expressed. In addition, expression of a pyruvate decarboxylase gene may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression. Moreover, a pyruvate decarboxylase encoding gene may be synthesized whose expression is low because rare codons are substituted for plentiful ones, and this gene substituted for the endogenous corresponding pyruvate decarboxylase encoding gene. Such a gene will produce the same polypeptide but at a lower rate. In addition, the synthesis or stability of the transcript may be lessened by mutation. Similarly the efficiency by which a protein is translated from mRNA may be modulated by mutation. All of these methods may be readily practiced by one skilled in the art making use of the known or identified sequences encoding pyruvate decarboxylase proteins.

DNA sequences surrounding a pyruvate decarboxylase coding sequence are also useful in some modification procedures and are available for yeasts such as for Saccharomyces cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. Additional examples of yeast genomic sequences include that of Yarrowia lipolytica, GOPIC #13837, and of Candida albicans, which is included in GPID #10771, #10701 and #16373. Other yeast genomic sequences can be readily found by one of skill in the art in publicly available databases.

In particular, DNA sequences surrounding a pyruvate decarboxylase coding sequence are useful for modification methods using homologous recombination. For example, in this method pyruvate decarboxylase gene flanking sequences are placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the pyruvate decarboxylase gene. Also partial pyruvate decarboxylase gene sequences and pyruvate decarboxylase gene flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the target pyruvate decarboxylase gene. In addition, the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the pyruvate decarboxylase gene without reactivating the latter. The site-specific recombination leaves behind a recombination site which disrupts expression of the pyruvate decarboxylase protein. The homologous recombination vector may be constructed to also leave a deletion in the pyruvate decarboxylase gene following excision of the selectable marker, as is well known to one skilled in the art.

Deletions may be made using mitotic recombination as described in Wach et al. ((1994) Yeast 10:1793-1808). This method involves preparing a DNA fragment that contains a selectable marker between genomic regions that may be as short as 20 bp, and which bound a target DNA sequence. This DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. The linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence (as described in Methods in Enzymology, v194, pp 281-301 (1991)).

Moreover, promoter replacement methods may be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh et al. ((2004) Cell 118(1):31-44) and in Example 12 herein.

In addition, pyruvate decarboxylase activity in any yeast cell may be disrupted using random mutagenesis, which is followed by screening to identify strains with reduced pyruvate decarboxylase activity. Using this type of method, the DNA sequence of the pyruvate decarboxylase encoding region, or any other region of the genome affecting expression of pyruvate carboxylase activity, need not be known.

Methods for creating genetic mutations are common and well known in the art and may be applied to the exercise of creating rmutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.

Chemical mutagenesis of yeast commonly involves treatment of yeast cells with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). These methods of mutagenesis have been reviewed in Spencer et al (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). Chemical mutagenesis with EMS may be performed as described in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer et al (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). Introduction of a mutator phenotype can also be used to generate random chromosomal mutations in yeast. Common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAG1, RAD18 or RAD51. Restoration of the non-mutator phenotype can be easily obtained by insertion of the wildtype allele. Collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced pyruvate decarboxylase activity.

Reduced Pyruvate Dehydrogenase Activity

Endogenous pyruvate dehydrogenase activity is in the yeast mitochondrion and catalyzes oxidative decarboxylation of pyruvate to form acetyl-CoA. Acetyl-CoA is used in the TCA cycle and in fatty acid biosynthesis. The pyruvate dehydrogenase enzyme is one enzyme of a multienzyme pyruvate dehydrogenase complex. Pyruvate dehydrogenase (EC 1.2.4.1) itself has alpha and beta subunits: PDA1 and PDB1, respectively, forming the E1 component. The complex includes an E2 core which has dihydrolipoamide acetyltransferase activity (EC 2.3.1.12) and E3 which has dihydrolipoamide dehydrogenase activity (EC1.8.1.4). E2 may be encoded by lat1 and E3 by lpd1. An additional complex protein is encoded by pdx1. Thus the pyruvate dehydrogenase complex may include PDA1, PDB1, Lat1, Lpd1, and Pdx1, or homologous proteins encoded by genes which may have alternative names in various yeasts.

Any of the genes encoding pyruvate dehydrogenase complex enzymes of yeast may be disrupted to reduce pyruvate dehydrogenase activity in a yeast cell to prepare a strain of one embodiment of the invention. Examples of yeast pyruvate dehydrogenase complex protein encoding genes that may be targeted for disruption are listed in Table 2 (SEQ ID NOs:82, 84, 86, 88, 90, 92, 94, 96, 98, 100). Other target genes, such as those encoding pyruvate dehydrogenase proteins having at least about 80-85%, 85%-9090%-95%, or at least about 98% sequence identity to the pyruvate dehydrogenases listed in Table 2 (SEQ ID NOs:83, 85, 87, 89, 91, 93, 95, 97, 99, 101) may be identified in the literature and in bioinformatics databases well known to the skilled person as described above. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature as described above for acetolactate synthase encoding sequences.

Accordingly it is within the scope of the invention to provide pyruvate dehydrogenase complex proteins having at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to any of the pyruvate dehydrogenase complex proteins disclosed herein (SEQ ID NO: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101). Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Genes encoding pyruvate dehydrogenase complex proteins may be disrupted in any yeast cell using genetic modification. Many methods for genetic modification of target genes are known to one skilled in the art and may be used to create the present yeast strains. Examples of methods that may be used are as described above for disruption of pyruvate decarboxylase encoding genes. In addition, pyruvate dehydrogenase activity in any yeast cell may be disrupted using random mutagenesis, which is followed by screening to identify strains with reduced pyruvate dehydrogenase activity. Using this type of method, the DNA sequence of the pyruvate dehydrogenase encoding region, or any other region of the genome affecting expression of pyruvate dehydrogenase activity, need not be known. Examples of methods are as described above for random mutagenesis of pyruvate decarboxylase.

Reduced glycerol-3-phosphate Dehydrogenase Activity

Endogenous NAD-dependent glycerol-3-phosphate dehydrogenase is a key enzyme in glycerol synthesis, converting dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate playing a major role in cellular oxidation of NADH. Yeast strains may have one or more genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD). In some yeasts, such as S. cerevisiae, S. pombe, and P. stipitis, GPD1 and GPD2 are functional homologs for NAD-dependent glycerol-3-phosphate dehydrogenase. Any of the genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase enzymes of yeast may be disrupted to reduce NAD-dependent glycerol-3-phosphate dehydrogenase activity in a yeast cell to prepare a strain of one embodiment of the invention. Examples of coding regions of yeast NAD-dependent glycerol-3-phosphate dehydrogenase protein encoding genes that may be targeted for disruption are listed in Table 2 (SEQ ID NOs:68, 70, 72, 74, 76, 80). Other target genes, such as those encoding NAD-dependent glycerol-3-phosphate dehydrogenase proteins having at least about 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to the NAD-dependent glycerol-3-phosphate dehydrogenases listed in Table 2 (SEQ ID NOs:69, 71, 73, 75, 77, 79, 81) may be identified in the literature and in bioinformatics databases well known to the skilled person. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature as described above for acetolactate synthase encoding sequences.

Accordingly it is within the scope of the invention to provide NAD-dependent glycerol-3-phosphate dehydrogenase proteins having at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to any of the NAD-dependent glycerol-3-phosphate dehydrogenase proteins disclosed herein (SEQ ID NO: 69, 71, 73, 75, 77, 79, 81). Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Genes encoding NAD-dependent glycerol-3-phosphate dehydrogenases may be disrupted in any yeast cell using genetic modification. Many methods for genetic modification of target genes are known to one skilled in the art and may be used to create the present yeast strains. In addition, random mutagenesis and screening may be used to disrupt expression of NAD-dependent glycerol-3-phosphate dehydrogenases. Examples of genetic modification and random mutagenesis methods that may be used are as described above for disruption of pyruvate decarboxylase encoding genes.

Yeast Cells with Enhanced Pyruvate Conversion to Acetolactate

The characteristics of the yeast strains disclosed herein may be made in any yeast host that is amenable to genetic manipulation. Examples include yeasts of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis and Yarrowia lipolytica. Most suitable is Saccharomyces cerevisiae.

For maximal production of some products such as 2,3-butanediol, isobutanol, 2-butanone, or 2-butanol the yeast strains used as production hosts preferably have enhanced tolerance to the produced chemical, and have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural.

Product Biosynthesis in Enhanced Pyruvate to Acetolactate Conversion Strain

Any product that has acetolactate as a pathway intermediate may be produced with greater effectiveness in a yeast strain disclosed herein having enhanced conversion of pyruvate to acetolactate. A list of such products includes, but is not limited to, valine, leucine, isoamyl alcohol, 2,3-butanediol, 2-butanone, 2-butanol, and isobutanol.

Biosynthesis of valine includes steps of acetolactate conversion to 2,3-dihydroxy-isovalerate by acetohydroxyacid reductoisomerase (ILV5), conversion of 2,3-dihydroxy-isovalerate to 2-keto-isovalerate by dihydroxy-acid dehydratase (ILV3), and conversion of 2-keto-isovalerate to valine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). Biosynthesis of leucine includes the same steps to 2-keto-isovalerate, followed by conversion of 2-keto-isovalerate to alpha-isopropylmalate by alpha-isopropylmalate synthase (LEU9, LEU4), conversion of alpha-isopropylmalate to beta-isopropylmalate by isopropylmalate isomerase (LEU1), conversion of beta-isopropylmalate to alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally conversion of alpha-ketoisocaproate to leucine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). Increased conversion of pyruvate to acetolactate will increase flow to these pathways, particularly if one or more enzymes of the pathway is overexpressed to pull acetolactate into the pathway. Thus it is desired for production of valine or leucine to overexpress at least one of the enzymes in these described pathways.

Biosynthesis of isoamyl alcohol includes steps of leucine conversion to alpha-ketoisocaproate by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1), conversion of alpha-ketoisocaproate to 3-methylbutanal by ketoisocaproate decarboxylase (THI3) or decarboxylase ARO10, and finally conversion of 3-methylbutanal to isoamyl alcohol by an alcohol dehydrogenase such as ADH1 or SFA1. Thus further production of isoamyl alcohol benefits from increased production of leucine or the alpha-ketoisocaproate intermediate by overexpression of one or more enzymes in biosynthetic pathways for these chemicals. In addition, one or both enzymes for the final two steps may be overexpressed.

Biosynthetic pathways starting with a step of converting pyruvate to acetolactate for synthesis of isobutanol are disclosed in commonly owned and co-pending US Patent Application publication US 20070092957 A1, which is herein incorporated by reference. A diagram of the disclosed isobutanol biosynthetic pathways is provided in FIG. 2. Production of isobutanol in a strain disclosed herein benefits from increased availability of acetolactate. As described in US 20070092957 A1, steps in an example isobutanol biosynthetic pathway using acetolactate include conversion of:

acetolactate to 2,3-dihydroxyisovalerate (FIG. 2 pathway step b) as catalyzed for example by acetohydroxy acid isomeroreductase;

2,3-dihydroxyisovalerate to α-ketoisovalerate (FIG. 2 pathway step c) as catalyzed for example by acetohydroxy acid dehydratase;

α-ketoisovalerate to isobutyraldehyde (FIG. 2 pathway step d) as catalyzed for example by branched-chain α-keto acid decarboxylase; and

isobutyraldehyde to isobutanol (FIG. 2 pathway step e) as catalyzed for example by branched-chain alcohol dehydrogenase.

Genes that may be used for expression of these enzymes, as well as those for two additional isobutanol pathways, are described in US 20070092957 A1, and additional genes that may be used can be identified by one skilled in the art. The preferred use in all three pathways of ketol-acid reductoisomerase (KARI) enzymes with particularly high activities are disclosed in commonly owned and co-pending US Patent Application Publication #20080261230. Examples of high activity KARIs disclosed therein are those from Vibrio cholerae (DNA: SEQ ID NO:39; protein SEQ ID NO:40), Pseudomonas aeruginosa PAO1, (DNA: SEQ ID NO:41; protein SEQ ID NO:42), and Pseudomonas fluorescens PF5 (DNA: SEQ ID NO:43; protein SEQ ID NO:44).

Useful for the last step of converting isobutyraldehyde to isobutanol is a new butanol dehydrogenase isolated from an environmental isolate of a bacterium identified as Achromobacter xylosoxidans that is disclosed in commonly owned and co-pending U.S. patent application Ser. No. 12/430,356 (DNA: SEQ ID NO:35, protein SEQ ID NO:36).

Additionally described in US 20070092957 A1 are construction of chimeric genes and genetic engineering of yeast, exemplified by Saccharomyces cerevisiae, for isobutanol production using the disclosed biosynthetic pathways.

Biosynthetic pathways starting with a step of converting pyruvate to acetolactate for synthesis of 2-butanone and 2-butanol are disclosed in commonly owned and co-pending US Patent Application publications US 20070259410A1 and US 20070292927A1, which are herein incorporated by reference. A diagram of the disclosed 2-butanone and 2-butanol biosynthetic pathways is provided in FIG. 3. 2-Butanone is the product made when the last depicted step of converting 2-butanone to 2-butanol is omitted. Production of 2-butanone or 2-butanol in a strain disclosed herein benefits from increased availability of acetolactate. As described in US 20070292927A1, steps in an example biosynthetic pathway using acetolactate include conversion of:

-   -   acetolactate to acetoin (FIG. 3 step b) as catalyzed for example         by acetolactate decarboxylase;     -   acetoin to 2,3-butanediol (FIG. 3 step i) as catalyzed for         example by butanediol dehydrogenase;     -   2,3-butanediol to 2-butanone (FIG. 3 step j) as catalyzed for         example by diol dehydratase or glycerol dehydratase; and     -   2-butanone to 2-butanol (FIG. 3 step f) as catalyzed for example         by butanol dehydrogenase.

Genes that may be used for expression of these enzymes are described in US 20070292927A1. The use in this pathway in yeast of the butanediol dehydratase from Roseburia inulinivorans, RdhtA, (protein SEQ ID NO:32, coding region SEQ ID NO:31) is disclosed in commonly owed and co-pending U.S. patent application Ser. No. 12/111,359. This enzyme is used in conjunction with the butanediol dehydratase reactivase from Roseburia inulinivorans, RdhtB, (protein SEQ ID NO:34, coding region SEQ ID NO: 33). This butanediol dehydratase is desired in many hosts because it does not require coenzyme B₁₂.

Useful for the last step of converting 2-butanone to 2-butanol is a new butanol dehydrogenase isolated from an environmental isolate of a bacterium identified as Achromobacter xylosoxidans that is disclosed in commonly owned and co-pending U.S. patent application Ser. No. 12/430,356 (DNA: SEQ ID NO:35, protein SEQ ID NO:36).

Additionally described in U.S. patent application Ser. No. 12/111,359 are construction of chimeric genes and genetic engineering of yeast for 2-butanol production using the US 20070292927A1 disclosed biosynthetic pathway. 2,3-butanediol is an intermediate in this 2-butanol pathway and the steps in its synthesis are also described above.

Fermentation Media

Yeasts disclosed herein may be grown in fermentation media for production of a product having acetolactate as an intermediate. Fermentation media must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for production of the desired product.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C. to about 37° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.

Suitable pH ranges for the fermentation are between pH 3.0 to pH 7.5, where pH 4.5.0 to pH 6.5 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

The amount of butanol produced in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.

Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 1-butanol production.

Methods for Butanol Isolation from the Fermentation Medium

Bioproduced butanol may be isolated from the fermentation medium using methods known in the art. For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. Because butanol forms a low boiling point, azeotropic mixture with water, distillation can only be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see for example Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol. In this method, the butanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.

The butanol may also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent.

Distillation in combination with adsorption may also be used to isolate butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation may be used to isolate and purify the butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987), and by Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. Microbial strains were obtained from The American Type Culture Collection (ATCC), Manassas, Va., unless otherwise noted. The oligonucleotide primers used in the following Examples are given in Table 3. All the oligonucleotide primers were synthesized by Sigma-Genosys (Woodlands, Tex.) or Integrated DNA Technologies (Coralsville, Iowa).

Synthetic complete medium is described in Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

GC Method

The GC method utilized an HP-InnoWax column (30 m×0.32 mm ID, 0.25 μm film) from Agilent Technologies (Santa Clara, Calif.). The carrier gas was helium at a flow rate of 1 ml/min measured at 150° C. with constant head pressure; injector split was 1:10 at 200° C.; oven temperature was 45° C. for 1 min, 45° C. to 230° C. at 10° C./min, and 230° C. for 30 sec. FID detection was used at 260° C. with 40 ml/min helium makeup gas. Culture broth samples were filtered through 0.2 μM spin filters before injection. Depending on analytical sensitivity desired, either 0.1 μl or 0.5 μl injection volumes were used. Calibrated standard curves were generated for the following compounds: ethanol, isobutanol, acetoin, meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. Analytical standards were also utilized to identify retention times for isobutyraldehyde, isobutyric acid, and isoamyl alcohol.

HPLC

Analysis for fermentation by-product composition is well known to those skilled in the art. For example, one high performance liquid chromatography (HPLC) method utilizes a Shodex SH-1011 column with a Shodex SH-G guard column (both available from Waters Corporation, Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H₂SO₄ as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol retention time is 47.6 minutes.

For Example 15, isobutanol concentration in the aqueous phase was measured by HPLC (Waters Alliance Model, Milford, Mass. or Agilent 1100 Series, Santa Clara, Calif.) using a Shodex sugar SH1011 column, 8.0 mm×300 mm, (Showa Denko K.K., Kanagawa, Japan (through Thompson Instruments, Clear Brook, Va.)) using 0.01 N aqueous sulfuric acid, isocratic, as the eluant. The sample was passed through a 0.2 μm syringe filter (PALL GHP membrane) into an HPLC vial. The HPLC run conditions were as follows:

Injection volume: 10 μL

Flow rate: 0.80 mL/minute

Run time: 32 minutes

Column Temperature: 50° C.

Detector: refractive index

Detector temperature: 40° C.

UV detection: 210 nm, 4 nm bandwidth

After the run, concentration in the sample was determined from a standard curves for isobutanol. The retention time was 27.0 minutes.

The meaning of abbreviations is as follows: “s” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s)”, “g” means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density, “OD₆₀₀” means the optical density measured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, % v/v” means volume/volume percent, “wt/%” means percent by weight, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography. The term “molar selectivity” is the number of moles of product produced per mole of sugar substrate consumed and is reported as a percent.

Example 1 Disruption of Pyruvate Decarboxylase Genes

The purpose of this example is to describe disruption of pyruvate decarboxylase genes in S. cerevisiae by chromosomal deletion of genes encoding the three major isozymes: PDC1, PDC5, and PDC6.

The PDC1 gene, encoding a first isozyme of pyruvate decarboxylase, was disrupted by insertion of a LEU2 marker cassette by homologous recombination, which completely removed the endogenous PDC1 coding sequence. The LEU2 marker in pRS425 (ATCC No. 77106) was PCR-amplified from plasmid DNA using Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) using primers PDC1::LEU2-F and PDC1::LEU2-R, given as SEQ ID NOs:102 and 103, which generated a 2.0 kb PCR product. The PDC1 portion of each primer was derived from the 5′ region upstream of the PDC1 promoter and 3′ region downstream of the transcriptional terminator, such that integration of the LEU2 marker results in replacement of the pdc1 coding region. The PCR product was transformed into S. cerevisiae BY4741 (ATCC #201388) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking leucine and supplemented with 2% glucose at 30° C. Transformants were screened by PCR using primers 112590-30E and 112590-30F, given as SEQ ID NOs:104 and 105, to verify integration at the PDC1 chromosomal locus with replacement of the PDC1 coding region. The identified correct transformants have the genotype: BY4741 pdc1::LEU2.

The PDC5 locus encoding a second isozyme of pyruvate decarboxylase was deleted by gene disruption. A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase and primers PDC5::KanMXF and PDC5::KanMXR, given as SEQ ID NOs:106 and 107, which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into BY4741 pdc1::LEU2 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175, given as SEQ ID NOs:108 and 109, respectively. The identified correct transformants have the genotype: BY4741 pdc1::LEU2 pdc5::kanMX4.

The PDC6 locus encoding a third isozyme of pyruvate decarboxylase was disrupted by insertion of a MET15 marker. The MET15 marker was PCR-amplified from plasmid pRS421 (ATCC No. 87475) using Phusion DNA polymerase and primers 112590-46A and 112590-46B, given as SEQ ID NOs: 110 and 111, respectively. The PDC6 portion of each primer was derived from the 5′ region upstream of the PDC6 promoter and 3′ region downstream of the coding region, such that integration of the MET15 marker results in replacement of the PDC6 coding region. The PCR product was transformed into BY4741 pdc1::LEU2 pdc5::kanMX4 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking methionine and supplemented with 1% ethanol at 30° C. Transformants were screened by PCR to verify correct chromosomal integration at the PDC6 locus with replacement of the PDC6 coding region using primers 112590-34E and 112590-34F, given as SEQ ID NOs:112 and 113, respectively. The identified correct transformants have the genotype: BY4741 pdc1::LEU2 pdc5::kanMX4 pdc6::MET15.

Example 2 Engineering S. Cerevisiae Strains for Cytosolic Expression of Acetolactate Synthase and Deletion of Pyruvate Decarboxylase Genes

The purpose of this example is to describe the construction and introduction of acetolactate synthase genes for expression in the cytosol of a yeast strain that also has deletions of pyruvate decarboxylase genes PDC1 and PDC5. Two yeast promoters were independently used to control alsS gene expression—the glycolytic FBA promoter from the S. cerevisiae fructose 1,6-bisphosphate aldolase or the HIS3 promoter from the S. cerevisiae imidazoleglycerol-phosphate dehydratase gene.

Expression plasmid pRS426-FBAp-alsS was constructed via the following steps. The 1.7 kb alsS coding region fragment of pRS426::GPD::alsS::CYC was isolated by gel purification following BbvCI and PacI digestion. This plasmid has a chimeric gene containing the GPD1 promoter (SEQ ID NO:114), the alsS coding region from Bacillus subtilis (SEQ ID NO:3), and the CYC1 terminator (SEQ ID NO:115]) and was described in commonly owned and co-pending US Patent Publication # US20070092957 A1, Example 17 which is herein incorporated by reference. The ILV5 fragment from plasmid pRS426::FBA::ILV5::CYC, also described in US20070092957 A1, Example 17, was removed by restriction digestion with BbvCI and PacI and the remaining 6.6 kb vector fragment was gel purified. This vector has a chimeric gene containing the FBA promoter (SEQ ID NO:116) and CYC1 terminator bounding the coding region of the ILV5 gene of S. cerevisiae (SEQ ID NO:37). These two purified fragments were ligated overnight at 16° C. and transformed into E. coli TOP10 chemically competent cells (Invitrogen). Transformants were obtained by plating cells on LB Amp100 medium. Insertion of alsS into the vector was confirmed by restriction digestion pattern and PCR (primers N98SeqF1 and N99SeqR2, SEQ ID NOs:117 and 118).

A pdc1::FBAp-alsS-LEU2 disruption cassette was created by joining the FBAp-alsS segment from pRS426-FBAp-alsS to the LEU2 gene from pRS425 (ATCC No. 77106) by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pRS426-FBAp-alsS and pRS425 plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 112590-48A and 112590-30B through D, given as SEQ ID NOs:119, SEQ ID NOs:120-122. The outer primers for the SOE PCR (112590-48A and 112590-30D) contained 5′ and 3′ 50 bp regions homologous to regions upstream and downstream of the PDC1 promoter and terminator. The completed cassette PCR fragment was transformed into BY4741 (ATCC No. 201388) and transformants were maintained on synthetic complete media lacking leucine and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-30E and 112590-30F, given as SEQ ID NOs:104 and 105, to verify integration at the PDC1 locus with deletion of the PDC1 coding region. The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2.

A pdc1::HIS3p-alsS-LEU2 disruption cassette was created by amplifying the HIS3 promoter from pRS423 (ATCC No. 77104) with Phusion DNA polymerase and joining it to a PCR-amplified alsS-LEU2 cassette from strain BY4741 pdc1::FBAp-alsS-LEU2. Primers utilized for the PCR, 112590-48B through 112590-48D and 112590-45B, are given as SEQ ID NOs:161-163 and 164. The outer primers for the SOE PCR contained 5′ and 3′ 50 bp regions homologous to the regions upstream and downstream of the PDC1 promoter and terminator. The completed pdc1::HIS3p-alsS-LEU2 cassette was transformed into BY4741 and transformants were maintained on synthetic complete media lacking leucine and supplemented with 2% glucose at 30° C. (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using external primers 112590-30E and 112590-30F, given as SEQ ID NOs:104 and 105, to verify integration at the PDC1 locus with deletion of the PDC1 coding region. The correct transformants have the genotype: BY4741 pdc1::HIS3p-alsS-LEU2.

The PDC5 locus encoding the second isozyme of pyruvate decarboxylase was deleted by gene disruption. The pdc5::kanMX4 cassette was PCR-amplified using Phusion DNA polymerase from strain YLR134W chromosomal DNA (ATCC No. 4034091) using primers PDC5::KanMXF and PDC5::KanMXR, given as SEQ ID NOs:106 and 107, which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′region downstream of the coding region, such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into BY4741 pdc1::FBAp-alsS-LEU2 and BY4741 pdc1::HIS3p-alsS-LEU2 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) with selection on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct chromosomal integration using primers PDC5kofor and N175, given as SEQ ID NOs:108 and 109, respectively. The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4.

Example 3 Vector Construction for the Production of Butanediol

The purpose of this example is to describe the construction of vectors for the expression of acetolactate decarboxylase, butanediol dehydrogenase, and, optionally, acetolactate synthase and/or secondary alcohol dehydrogenase activity in the cytosol of yeast.

Construction of pRS423::CUP1-alsS+FBA-budA

The budA gene, encoding acetolactate decarboxylase, was amplified from genomic DNA prepared from Klebsiella pneumonia (ATCC #25955) using Phusion™ Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Inc.). The primers used (N579 and N580, provided as SEQ ID NOs:123 and 124) added sequence upstream of the start codon that was homologous to the yeast FBA promoter and sequence downstream of the stop codon that was homologous to the yeast ADH terminator. Plasmid pRS423::CUP1-alsS+FBA-ILV3, which has a chimeric gene containing the CUP1 promoter (SEQ ID NO:125), alsS coding region from Bacillus subtilis (SEQ ID NO:3), and CYC1 terminator (SEQ ID NO:115) as well as a chimeric gene containing the FBA promoter (SEQ ID NO:116), ILV3 coding region from S. cerevisiae (SEQ ID NO:45), and ADH1 terminator (SEQ ID NO:126) (described in commonly owned and co-pending US Patent Publication # US20070092957 A1, Example 17) was restriction digested with NcoI and PmlI to remove the ILV3 coding region. The 11.1 kb vector band was gel purified. Approximately 1 μg of cut vector DNA was combined with 1 μg of the budA PCR product and transformed into S. cerevisiae strain BY4741. The insert and vector were combined by homologous recombination in vivo to form a circular vector (also known as “gap repair cloning”; described in Ma et al. (1987) Genetics 58:201-216) that allows retention of the selectable marker (in this case, HIS3). Transformants were selected on synthetic complete medium lacking histidine. Colonies were patched to a new plate and cells from these patches were used to prepare plasmid DNA (Zymoprep™ Yeast Plasmid Miniprep Kit, Zymo Research). PCR was used to screen plasmids for the presence of alsS (primers N98SeqF1 and N99SeqR2, SEQ ID NOs: 117 and 118) and for proper insertion of budA (N160SeqF1 and N84SeqR2, SEQ ID NOs:127 and 128).

Construction of pRS426::FBA-budC

The budC coding region for butanediol dehydrogenase was amplified from genomic DNA prepared from Klebsiella pneumonia (ATCC #25955) using Phusion™ Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Inc.). The primers used (N581 and N582, provided as SEQ ID NOs:129 and 130) added sequence upstream of the start codon that was homologous to the yeast FBA promoter and sequence downstream of the stop codon that was homologous to the yeast CYC1 terminator. The plasmid pRS426::FBA::alsS (described in Example 2, also called pRS426-FBAp-alsS) was digested with BbvCI and PacI to release an alsS fragment. The remaining linear vector was gel purified. Approximately 1 μg of vector was combined with 1 μg of budC PCR product and transformed into BY4741 to obtained gap repair clones (see above). Transformants were selected on synthetic complete medium lacking uracil. Plasmids were prepared from patches of 5 transformant colonies. The presence of FBA-budC was screened using PCR with primers N160SeqF1 and N582 (SEQ ID NOs:127 and 130).

Construction of pRS423::FBA-budC+FBA-budA

The pRS423::CUP1-alsS+FBA-budA vector described above was digested with SacII and MluI to remove CUP1-alsS. SacII/MluI digestion was also used to isolate FBA-budC from pRS426::FBA-budC (see above). The appropriate fragments (7.6 kb vector fragment and 1.6 kb FBA-budC fragment) were gel purified, ligated and transformed into E. coli TOP10 competent cells (Invitrogen). Transformant colonies were screened by PCR to confirm incorporation of the budC fragment using primers N581 and N582 (SEQ ID NOs:129 and 130).

Construction of pRS425::GPM-sadB

A DNA fragment encoding a butanol dehydrogenase (protein of SEQ ID NO:36) from Achromobacter xylosoxidans (disclosed in commonly owned and co-pending US Patent Application CL3926) was cloned. The coding region of this gene called sadB for secondary alcohol dehydrogenase (SEQ ID NO:35) was amplified using standard conditions from A. xylosoxidans genomic DNA, prepared using a Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5500A) following the recommended protocol for gram negative organisms using forward and reverse primers N473 and N469 (SEQ ID NOs:131 and 132), respectively. The PCR product was TOPO-Blunt cloned into pCR4 BLUNT (Invitrogen) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-1 cells. Plasmid was subsequently isolated from four clones, and the sequence verified.

The sadB coding region was PCR amplified from pCR4Blunt::sadB. PCR primers contained additional 5′ sequences that would overlap with the yeast GPM1 promoter and the ADH terminator (N583 and N584, provided as SEQ ID NOs:133 and 134). The PCR product was then cloned using “gap repair” methodology in Saccharomyces cerevisiae (Ma et al. ibid) as follows. The yeast-E. coli shuttle vector pRS425::GPM::kivD::ADH which contains the GPM promoter (SEQ ID NO:135), kivD coding region from Lactococcus lactis (SEQ D NO:47), and ADH1 terminator (SEQ ID NO:126) (described in commonly owned and co-pending US Patent Publication # US20070092957 A1, Example 17) was digested with BbvCI and PacI restriction enzymes to release the kivD coding region. Approximately 1 μg of the remaining vector fragment was transformed into S. cerevisiae strain BY4741 along with 1 μg of sadB PCR product. Transformants were selected on synthetic complete medium lacking leucine. The proper recombination event, generating pRS425::GPM-sadB, was confirmed by PCR using primers N142 and N459 (SEQ ID NOs:136 and 137).

Construction of pRS426::FBA-budC+GPM-sadB

The GPM-sadB-ADH promoter-gene-terminator cassette was transferred to pRS426 (ATCC No. 77107), a yeast-E. coli shuttle vector carrying the URA3 selection marker, by gap repair cloning. The cassette was isolated from pRS425::GPM-sadB by digestion with SalI and SacII, and the pRS426 vector was linearized with BamHI prior to ligation. The resulting vector, pRS426::GPM-sadB was confirmed by PCR using primers N142 and N459 (SEQ ID NOs:136 and 137). In order to add the budC gene encoding acetoin reductase from Klebsiella pneumonia to this vector, a fragment containing budC was excised from pRS423::FBA-budC+FBA-budA using SphI and SapI.

For construction of pRS423::FBA-budC+FBA-budA, the pRS423::CUP1-alsS+FBA-budA vector described above was digested with SacII and MluI to remove CUP1-alsS. SacII/MluI digestion was also used to isolate FBA-budC from pRS426::FBA-budC (described above). The appropriate fragments (7.6 kb vector fragment and 1.6 kb FBA-budC fragment) were gel purified, ligated and transformed into E. coli TOP10 competent cells (Invitrogen). Transformant colonies were screened by PCR to confirm incorporation of the budC fragment using primers N581 and N582 (SEQ ID NOs:129 and 130).

The SphI-SapI budC fragment from pRS423::FBA-budC+FBA-budA carries portions of the vector upstream of the FBA promoter as well as part of the ADH terminator to allow for cloning by gap repair cloning into the pRS426::GPM-sadB vector that was linearized with SacII. Transformants resulting from this cloning were plated on medium lacking uracil to select for recombination of the two linear sequences. The resulting vector, pRS426::FBA-budC+GPM-sadB was confirmed by PCR using primers N581 and N582 (SEQ ID NOs:129 and 130).

Example 4 Production of Butanediol

The purpose of this example is to describe the production of butanediol in a yeast strain. The yeast strain comprises deletions of PDC1 and PDC5, genes encoding two isozymes of pyruvate decarboxylase, and constructs for heterologous expression of AlsS (acetolactate synthase), BudA (acetolactate decarboxylase), and BudC (butanediol dehydrogenase). Optionally, the yeast strain further comprises a construct for heterologous expression of SadB (secondary alcohol dehydrogenase).

Strain Construction

Plasmids pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC were introduced into BY4741 Δpdc1::FBA-alsS Δpdc5::kanMX4 and into BY4741 Δpdc1::HIS3-alsS Δpdc5::kanMX4 by standard PEG/lithium acetate-mediated transformation methods. Plasmids pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB were introduced into BY4741 Δpdc1::HIS3-alsS Δpdc5::kanMX4. In all cases, transformants were selected on synthetic complete medium lacking glucose, histidine and uracil. Ethanol (1% v/v) was used as the carbon source. After three days, transformants were patched to synthetic complete medium lacking histidine and uracil. This medium contained both 2% glucose and 1% ethanol as carbon sources. The resulting strains were further examined, as described below.

Production of BDO from Glucose in Shake Flasks

The strains BY4741 Δpdc1::FBA-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC (Strain 1 in Table 3) and BY4741 Δpdc1::HIS3-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC (Strain 2 in Table 3) were grown in synthetic complete medium without uracil or histidine, supplemented with 0.5% (v/v) ethanol. The strains were grown in vented flasks or sealed vials as listed in Table 3, with agitation (225 rpm) at 30° C. After 48 hours filtered culture medium was analyzed by HPLC using the sugar column method described in General Methods.

TABLE 3 Butanediol (BDO) production in engineered yeast strains BDO* BDO Molar Glycerol Molar Strain Culture Condition Titer, mM Selectivity Selectivity 1 75 ml culture in 76 0.64 0.47 125 ml vented flask 1 40 ml culture in 29 0.58 0.54 50 ml sealed vial 2 40 ml culture in 52 0.64 0.51 50 ml sealed vial *Butanediol (BDO) refers to the sum of meso-2,3-butanediol, (2S,3S)-(+)-2,3-butanediol and (2R,3R)-(−)-2,3-butanediol. The latter two forms are also referred to as ±-butanediol. Molar selectivity is moles product/moles glucose consumed. Production of BDO from Glucose in Fermenters

The strain BY4741 Δpdc1::FBA-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC (Strain 1 in Table 3) was grown in 2 L baffled shake flasks containing 0.4 L medium at 30° C. with shaking at 200 RPM. The medium contained per L: 6.7 g yeast nitrogen base without amino acids (DIFCO, product #291940); 0.1 g L-leucine; 0.02 g L-tryptophan; 1.4 g yeast synthetic drop-out medium supplements without histidine, leucine, tryptophan and uracil (Sigma, product #Y2001); 20 g D-glucose; and 10 mL ethanol. When the cells in the flask reached an OD₆₀₀ of 2.9, 60 mL aliquots were used to inoculate fermenters.

One liter fermenters were prepared with 540 mL of medium containing (per L): 6.7 g yeast nitrogen base without amino acids (DIFCO, product #291940); 0.2 g L-leucine; 0.04 g L-tryptophan; 2.8 g yeast synthetic drop-out medium supplements without histidine, leucine, tryptophan and uracil (Sigma, product #Y2001); and 10 mL ethanol. D-glucose (50% w/w) was added fed-batch so that concentration, initially at 30 g/L, varied between 30 and 5 g/L. Temperature was controlled at 30° C. and pH was maintained at pH 5.5 with the addition of either 50% (w/w) NaOH or 20% (w/v) H₃PO₄. Air was sparged at 0.3 standard liters per min without back pressure and the minimum stir speed was set to 100 rpm. dO was 100% initially and rpm was programmed to control dO at 30%, however, oxygen demand was low and the dO of all fermenters (Runs #1-3 in Table 5) remained in the 90% range throughout the phase of air sparging. In two fermenters (duplicate Runs #2-3), nitrogen sparge replaced air sparge at 35 hrs into the run. Over the course of the fermentations, samples were withdrawn for cell mass (OD₆₀₀), substrate utilization and by-product distribution measurements. Substrate and by-product concentrations were determined from HPLC analysis. The results are summarized in Table 4, below. Despite the difference in gas sparging between the fermenters, the results were not significantly different. Butanediol (BDO, sum of meso-butanediol and ±-butanediol) was produced at an average concentration of 229 mM with a molar selectivity of 0.63 (mole butanediol produced/moles glucose consumed). The molar selectivity obtained in shake flasks was identical to that obtained in fermenters.

TABLE 4 Fermentative production of butanediol (BDO) using E. coli strain BY4741 Δpdc1::FBA-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS + FBA-budA/pRS426::FBA-budC. Glucose meso- ±- Butanediol Time, consumed, Butanediol, Butanediol, Glycerol, Molar Run # hr OD₆₀₀ mM mM mM mM Selectivity Run #1 0 0.0 0 0 0 0 0.00 ″ 4 0.3 20 7 2 9 0.47 ″ 12 0.6 30 12 4 17 0.54 ″ 20 1.0 53 23 7 33 0.56 ″ 28 2.2 98 45 15 65 0.61 ″ 36 3.1 164 69 23 100 0.56 ″ 44 4.4 217 106 34 154 0.65 ″ 52 5.1 307 148 43 212 0.62 ″ 60 5.4 343 175 48 249 0.65 ″ 68 6.1 412 207 55 295 0.63 ″ 72 6.5 447 224 57 319 0.63 Run #2 0 0.0 0 0 0 0 0.00 ″ 4 0.3 19 7 2 10 0.52 ″ 12 0.6 32 12 4 17 0.50 ″ 20 1.0 58 22 7 32 0.50 ″ 28 2.3 106 44 15 64 0.56 ″ 36 3.5 170 70 23 101 0.55 ″ 44 4.7 234 109 34 157 0.61 ″ 52 5.9 327 154 44 217 0.61 ″ 60 5.9 367 184 50 258 0.64 ″ 68 6.8 441 219 57 304 0.63 ″ 72 7.2 467 237 60 328 0.64 Run #3 0 0.0 0 0 0 2 0.00 ″ 4 0.3 19 7 2 12 0.51 ″ 12 0.6 33 12 4 19 0.49 ″ 20 1.1 58 23 7 36 0.52 ″ 28 2.2 109 45 15 67 0.55 ″ 36 3.4 173 73 24 107 0.56 ″ 44 4.4 234 112 35 164 0.63 ″ 52 6.3 326 153 44 220 0.61 ″ 60 5.9 371 183 50 260 0.63 ″ 68 6.7 445 218 57 307 0.62 ″ 72 7.6 454 226 58 317 0.63 Coproduction of BDO and 2-Butanol from Sugar (Glucose or Sucrose) in the Presence of Absence of 2-Butanone

The strain BY4741 Δpdc1::HIS3-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC+GPM-sadB was grown in 50 ml culture medium in 125-ml vented flasks with agitation (225 rpm) at 30° C. Culture medium was synthetic complete medium without uracil or histidine, supplemented with 0.5% (v/v) ethanol with and without 2-butanone. When using 2-butanone, cultures were inoculated into serum vials (50 ml) containing 40 ml of aerobic medium (same medium as above but also containing 83 mM 2-butanone). Vials were then sealed with stoppers and crimps. Vials were also incubated at 30° C. with agitation. After 48 hours filtered culture medium was analyzed by HPLC (sugar column method).

The added 2-butanone serves as a substrate for production of 2-butanol by the sadB encoded secondary alcohol dehydrogenase, This reaction is used to balance reducing equivalents during BDO synthesis as follows:

The formation of BDO from glucose requires the concomitant production of a reducing equivalent (e.g. NADH): Glucose→BDO+NADH In the absence of 2-butanone, the reducing equivalent is absorbed by the production of a compound more reduced than glucose, e.g. glycerol: 0.5 Glucose+1 NADH→1 Glycerol In the presence of 2-butanone, the reducing equivalent is absorbed by the production of a more reduced derivative of 2-butanone, which acts as an electron sink: 2-Butanone+NADH→2-Butanol Thus, higher yield from glucose is obtained in the presence of the 2-butanone electron sink. Moreover, the capacity of S. cerevisiae, comprising Δpdc1::HIS3-alsS Δpdc5/pRS423::CUP1-alsS, to provide ≧0.86 C6 equivalents (from glucose or sucrose) to a product downstream of acetolactate was demonstrated, as given in Table 5.

TABLE 5 Production of butanediol and 2-butanol by engineered yeast strain. BDO BDO Glycerol 2-butanol % of Carbon Electron titer molar Molar titer theoretical source sink (mM) yield Yield (mM) yield BDO Glucose None 73 0.62 0.25 — 62 Glucose 2-butanone 102 0.90 0.11 78 90 Sucrose None 87 0.68 0.14 — 68 Sucrose 2-butanone 101 0.86 0.08 82 86

Data are averages of duplicate experiments.

Selectivity from sucrose is normalized to mol C6 sugar.

The strain BY4741 Δpdc1::LEU2 Δpdc5::kanMX4 (described in Example 1) was transformed with plasmids pRS423::CUP1-alsS-+FBA-budA and pRS426::FBA-budC+GPM-sadB (both plasmids described in Example 3) to produce the strain BY4741 Δpdc1::LEU2 Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC+GPM-sadB. This strain (Strain 4 in Table 6) contains alsS only on a plasmid, but is otherwise isogenic with Strain 3 of Table 6 which contains both chromosomal and plasmid copies of alsS. Strain 3 in Table 6 is BY4741 Δpdc1::HIS3-alsS Δpdc5::kanMX4/pRS423::CUP1-alsS+FBA-budA/pRS426::FBA-budC+GPM-sadB (described previously in Example 4). Strain 3 and two isolates of Strain 4 were grown in the presence of glucose and 2-butanone (MEK) as described above. The BDO molar yield is indistinguishable between strains.

TABLE 6 Production of butanediol and 2-butanol by engineered yeast strains. Glyc- 2- Elec- BDO BDO erol butanol % of Carbon tron titer molar Molar titer theoretical Strain source sink (mM) yield Yield (mM) yield BDO Strain 3 Glucose MEK 102 0.90 0.18 75 90 Strain 4, Glucose MEK 100 0.88 0.19 74 88 isolate 1 Strain 4, Glucose MEK 101 0.90 0.19 75 90 isolate 2

Example 5 (Prophetic) Production of 2-Butanol by Recombinant S. Cerevisiae Strain Additionally Expressing B₁₂-Independent Diol Dehydratase

A B₁₂-independent (S-adenosylmethionine (SAM)-dependent) butanediol dehydratase (SEQ ID NO:32) and its associated reactivase (SEQ ID NO:34) from the bacterium Roseburia inulinivorans are the topic of commonly owned and co-pending US Patent Application CL3893CIP. The sequences encoding these proteins (SEQ ID NOs:31 and 33, respectively), hereafter referred to as rdhtA and rdhtB, respectively, were synthesized as one DNA fragment (SEQ ID NO:138) by standard methods and cloned into an E. coli vector (by DNA2.0, Inc., Menlo Park, Calif.). The resulting clone was named pJ206::rdhtAB. The synthetic DNA fragment also contained a consensus ribosome binding site 5′ of the rdhtA coding region and terminal restriction sites recognized by BamHI (5′ end) and SalI (3′ end).

pJ206::rdhtAB was used as a PCR template to prepare separate RdhtA and RdhtB coding region fragments. The RdhtA coding region for the diol dehydratase was amplified by PCR using primers N695 and N696 (SEQ ID NOs:130 and 140). The RdhtB coding region for the diol dehydratase activase, was amplified by PCR using primers N697 and N698 (SEQ ID NOs:141 and 142). The two DNA fragments were combined with a dual terminator DNA fragment (SEQ ID NO:143) that has an ADH terminator (SEQ ID NO:126) and a CYC1 terminator (SEQ ID NO:115) adjacent to each other in opposing orientation using SOE PCR (Horton et al. (1989) Gene 77:61-68). The dual terminator fragment was isolated as a 0.6 kb fragment following PacI digestion of pRS426::FBA-ILV5+GPM-kivD (described in commonly owned and co-pending US Patent Publication #20070092957 A1, Example 17). The resulting 4 kb DNA fragment had the rdhtA and rdhtB coding regions in opposing orientation on either side of the dual terminator, with the 3′ end of each coding region adjacent to the dual terminator sequence. This DNA fragment was then cloned by gap repair methodology (Ma et al. (1987) Genetics 58:201-216) into the yeast shuttle vector pRS426::FBA-ILV5+GPM-kivD that was prepared by digestion with BbvCI to remove the ILV5 and kivD coding regions and dual terminator sequence between their 3′ ends.

pRS426::FBA-ILV5+GPM-kivD was described in commonly owned and co-pending US Patent Publication #20070092957 A1, Example 17. It contains in order: the FBA promoter (SEQ ID NO:116), the coding region of the ILV5 gene of S. cerevisiae (SEQ ID NO:37), the dual terminator fragment (SEQ ID NO:143), the kivD coding region from Lactococcus lactis (SEQ D NO:47), and the GPM promoter (SEQ ID NO:135), with the ILV5 and kivD coding regions in opposite orientation.

The resulting plasmid, pRS426::RdhtAB, contained the rdhtA gene under the control of the FBA promoter (SEQ ID NO:116) and the rdhtB gene under control of the GPM promoter (SEQ ID NO:135). The activity of the diol dehydratase in several of the yeast clones was confirmed by growing the yeast cells anaerobically in the presence of 1,2-propanediol and analyzing culture supernatants for the presence of propanol by GC or HPLC (as described in General Methods).

The FBA-RdhtA+GPM-RdhtB portion of pRS426::RdhtAB is then integrated into the yeast genome by homologous recombination, as follows. The region is amplified from the plasmid construct using primers N742 and N743 (SEQ ID NOs:144 and 145). Similarly, the URA3 marker is amplified from pRS426 (ATCC No. 77107) using primers N744a and N745a (SEQ ID NOs:146 and 147). These two DNA fragments are then combined using SOE PCR (Horton et al. (1989) Gene 77:61-68). The linear product is transformed into the BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX strain that was described in Example 2.

Transformants are obtained on medium lacking uracil. Integration at the former PDC5 locus (replacing the kanMX4 marker) is confirmed by PCR and by screening for geneticin sensitivity. Clones are tested for diol dehydratase activity as described above. The URA3 marker is recycled by passaging the clones in the presence of 5-fluororotic acid using standard yeast methods (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is used to confirm that the integrated RdhtA and RdhtB genes have been undisturbed by marker recycling.

The resulting strain, BY4741 pdc1::HIS3p-alsS pdc5::RdhtAB is then transformed with butanediol pathway plasmids pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB, that were described in Example 3. BDO and/or 2-butanol production is confirmed in the resulting transformants by HPLC or GC as described in General Methods. It is expected that cells grown with vigorous aeration on glucose produce only BDO and that cells grown under more anaerobic conditions convert some BDO to 2-butanol. Repeated passaging of the strains under anaerobic conditions may enhance production of 2-butanol, since the complete pathway does not result in net accumulation of NADH and therefore does not require loss of energy and carbon to glycerol formation.

Example 6 (Prophetic) Production of Isobutanol in Recombinant S. Cerevisiae [BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4]

The purpose of this prophetic example is to describe how to obtain isobutanol production in a yeast strain that is disrupted for pyruvate decarboxylase activities, and expresses cytosolic acetolactate synthase.

Construction of vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5-GPMp-kivD is described in US Patent Publication # US20070092957 A1, Example 17. pRS423::CUP1p-alsS+FBAp-ILV3 has a chimeric gene containing the CUP1 promoter (SEQ ID NO:125), the alsS coding region from Bacillus subtilis (SEQ ID NO:3), and CYC1 terminator (SEQ ID NO:115) as well as a chimeric gene containing the FBA promoter (SEQ ID NO:116), the coding region of the ILV3 gene of S. cerevisiae (SEQ ID NO:45), and the ADH1 terminator (SEQ ID NO:126). pHR81::FBAp-ILV5+GPMp-kivD is the pHR81 vector (ATCC #87541) with a chimeric gene containing the FBA promoter, the coding region of the ILV5 gene of S. cerevisiae (SEQ ID NO:37), and the CYC1 terminator as well as a chimeric gene containing the GPM promoter (SEQ ID NO:135), the coding region from kivD gene of Lactococcus lactis (SEQ ID NO:47), and the ADH1 terminator. pHR81 has URA3 and leu2-d selection markers.

The PDC6 locus encoding a third isozyme of pyruvate decarboxylase is disrupted by insertion of a MET15 marker in BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 (described in Example 2) using the method described in Example 1. The correct transformants are identified as having the genotype: BY4741 pdc1::LEU2 pdc5::kanMX4 pdc6::MET15.

Plasmid vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5+GPMp-kivD are transformed into strain BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 or BY4741 pdc1::LEU2 pdc5::kanMX4 pdc6::MET15 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol. Aerobic cultures are grown in 250 ml flasks containing 50 ml synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose and 0.5% ethanol in an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Low oxygen cultures are prepared by adding 45 mL of medium to 60 mL serum vials that are sealed with crimped caps after inoculation and kept at 30° C. Approximately 24 h and 48 h after induction with 0.03 mM CuSO₄ (final concentration), an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection and GC(HP-Innowax, 0.32 mm×0.25 μm×30 m (Agilent Technologies, Inc., Santa Clara, Calif.) with flame ionization detection (FID) for isobutanol content. Isobutanol is detected.

Example 7 (Prophetic) Disruption of Glycerol Formation in a S. Cerevisiae Strain with Deleted Genes Encoding Pyruvate Decarboxylase and Cytosolic Expression of Acetolactate Synthase

The purpose of this prophetic example is to describe how to disrupt glycerol formation in a yeast strain that is also disrupted in pyruvate decarboxylase genes, and contains a cassette for expression of cytosolic acetolactate synthase.

GPD1 encodes an NAD-dependent glycerol-3-phosphate dehydrogenase which is a key enzyme in glycerol synthesis and plays a major role in cellular oxidation of NADH. A gpd1::URA3 disruption cassette is constructed by PCR amplification of the URA3 marker from pRS426 (ATCC No. 77107) with primers 112590-T8 and 112590-T9, given as SEQ ID NOs:148 and 149. These primers create a 1.4 kb URA3 PCR product that contains 70 bp 5′ and 3′ extensions identical to sequences upstream and downstream of the GPD1 chromosomal locus for homologous recombination. The PCR product is transformed into BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) with selection on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants are screened by PCR using primers 112590-T4 and 112590-T10, given as SEQ ID NOs:150 and 151, to verify integration at the correct site and disruption of GPD1. The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 gpd1::URA3 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 gpd1::URA3. The URA3 marker is disrupted if desired by plating on 5-fluorootic acid (5FOA; Zymo Research, Orange, Calif.) using standard yeast techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) producing strains BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 and BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1.

GPD2 encodes an NAD-dependent glycerol-3-phosphate dehydrogenase and is a functional homolog of GPD1. A gpd2::URA3 disruption cassette is constructed by PCR amplification of the URA3 marker from pRS426 (ATCC No. 77107) with primers 112590-T11 and 112590-T12, given as SEQ ID NOs:152 and 153. These primers create a 1.4 kb URA3 PCR product that contains 70 bp 5′ and 3′ extensions identical to sequences upstream and downstream of the GPD2 chromosomal locus for homologous recombination. The PCR product is transformed into BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) with selection on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants are screened by PCR using primers 112590-T13 and 112590-T4, given as SEQ ID NOs:154 and 150, to verify integration at the correct site and disruption of GPD2. The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 gpd2::URA3 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 gpd2::URA3. The URA3 marker is disrupted by plating on 5-fluorootic acid (5FOA; Zymo Research, Orange, Calif.) using standard yeast techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) producing strains BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1Δgpd2 and BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2.

Example 8 (Prophetic) Production of Isobutanol in Recombinant S. Cerevisiae [BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1Δgpd2]

The purpose of this prophetic example is to describe how to obtain isobutanol production in a yeast strain that is disrupted for pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase activities, and expresses cytosolic acetolactate synthase.

Plasmid vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5+GPMp-kivD (see Example 6) are transformed into strain BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1Δgpd2 (described in Example 7) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol. Aerobic cultures are grown in 250 ml flasks containing 50 ml synthetic complete media lacking histidine and uracil, and supplemented with 2% glucose and 0.5% ethanol in an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Low oxygen cultures are prepared by adding 45 mL of medium to 60 mL serum vials that are sealed with crimped caps after inoculation and kept at 30° C. Approximately 24 h and 48 h after induction with 0.03 mM CuSO₄ (final concentration), an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection) and GC(HP-Innowax, 0.32 mm×0.25 μm×30 m (Agilent Technologies, Inc., Santa Clara, Calif.) with flame ionization detection (FID) for isobutanol content. Isobutanol is detected.

Example 9 (Prophetic) Increasing Pyruvate Accessibility by Disruption of Pyruvate Dehydrogenase in a S. Cerevisiae Strain with Deleted Genes Encoding Pyruvate Decarboxylase and Cytosolic Expression of Acetolactate Synthase

The purpose of this prophetic example is to describe how to increase pyruvate accessibility by disrupting pyruvate dehydrogenase in a yeast strain that is also disrupted for pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase, and contains a cassette for expression of cytosolic acetolactate synthase.

PDA1 encodes the alpha subunit of pyruvate dehydrogenase. Pyruvate dehydrogenase, consisting of alpha (Pda1p) and beta (Pdb1p) subunits, is the E1 component of the large multienzyme pyruvate dehydrogenase complex. Cells lacking PDA1 are viable but lack pyruvate dehydrogenase activity, show slower growth on glucose, and exhibit increased formation of petites that lack mitochondrial DNA. A pda1::URA3 disruption cassette is constructed by PCR amplification of the URA3 marker from pRS426 (ATCC No. 77107) with primers 112590-T1 and 112590-T2, given as SEQ ID NOs:155 and 156. These primers create a 1.4 kb URA3 PCR product that contains 70 bp 5′ and 3′ extensions identical to sequences upstream and downstream of the PDA1 chromosomal locus for homologous recombination. The PCR product is transformed into BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and maintained on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants are screened by PCR using primers 112590-T3 and 112590-T4, given as SEQ ID NOs:157 and 150, to verify integration at the correct site and disruption of PDA1. The absence of pyruvate dehydrogenase activity could also be confirmed by measuring enzyme activity as described by Neveling et al. (J. Bacteriol. 180(6):1540-8). The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 pda1::URA3 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 pda1::URA3. The URA3 marker is disrupted if desired by plating on 5-fluorootic acid (5FOA; Zymo Research, Orange, Calif.) using standard yeast techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) producing strains BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 Δpda1 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 Δpda1

Example 10 (Prophetic) Production of Isobutanol in Recombinant S. Cerevisiae [BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1Δgpd2 Δpda1]

The purpose of this prophetic example is to describe how to obtain isobutanol production in a yeast strain that is disrupted for pyruvate decarboxylase, glycerol-3-phosphate dehydrogenase, and pyruvate dehydrogenase activities, and also expresses cytosolic acetolactate synthase.

Plasmid vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5+GPMp-kivD (see Example 6) are transformed into strain BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1Δgpd2 Δpda1 (see Example 9) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). and maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol. Aerobic cultures are grown in 250 ml flasks containing 50 ml synthetic complete media (minus histidine and uracil) supplemented with 2% glucose and 0.5% ethanol in an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Low oxygen cultures are prepared by adding 45 mL of medium to 60 mL serum vials that are sealed with crimped caps after inoculation and kept at 30° C. Approximately 24 h and 48 h after induction with 0.03 mM CuSO₄ (final concentration), an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection) and GC(HP-Innowax, 0.25 mm×0.2 μm×25 m (Agilent Technologies, Inc., Santa Clara, Calif.) with flame ionization detection (FID) for isobutanol content.

Example 11 (Prophetic) Increasing Pyruvate Accessibility by Suppression of Pyruvate Dehydrogenase in a S. Cerevisiae Strain with Deleted Genes Encoding Pyruvate Decarboxylase and Glycerol-3-Phosphate Dehydrogenase, and Cytosolic Expression of Acetolactate Synthase

The purpose of this prophetic example is to describe how to increase pyruvate accessibility by disrupting pyruvate dehydrogenase in a yeast strain that is also disrupted for pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase, and contains a cassette for overexpression of acetolactate synthase.

PDA1 encodes the alpha subunit of pyruvate dehydrogenase. Pyruvate dehydrogenase, consisting of alpha (Pda1p) and beta (Pdb1p) subunits, is the E1 component of the large multienzyme pyruvate dehydrogenase complex. Cells lacking PDA1 are viable but lack pyruvate dehydrogenase activity, show slower growth on glucose, and exhibit increased formation of petites that lack mitochondrial DNA. To suppress expression of PDA1, we will substitute the endogenous PDA1 promoter with the GAL1 promoter which is repressed when glucose is present in the media.

A URA3::GAL1p-PDA1 integration cassette is constructed by SOE PCR. The URA3 marker is amplified from pRS426 (ATCC No. 77107) with primers 112590-T1 and 112590-T5, given as SEQ ID NOs:155 and 158. The GAL1 promoter is PCR-amplified from plasmid pYES2 (Invitrogen, Carlsbad, Calif.) with primers 112590-T6 and 112590-T7, given as SEQ ID NOs:159 and 160. The two PCR products are fused together by SOE PCR and amplified with external primers 112590-T1 and 112590-T7, yielding a 1.8 kb PCR product. These primers add 5′ and 3′ extensions identical to sequences upstream of the PDA1 locus and to the coding sequence of PDA1 for homologous recombination. The PCR product is transformed into BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and maintained on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants are screened by PCR using primers 112590-T3 and 112590-T4, given as SEQ ID NOs:157 and 150, to verify integration at the correct site and disruption of PDA1. The suppression of pyruvate dehydrogenase activity is confirmed by measuring enzyme activity as described by Neveling, et al. [J. Bacteriol. 180(6):1540-8]. The correct transformants have the genotype: BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 URA3::GAL1p-PDA1 or BY4741 pdc1::HIS3p-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 URA3::GAL1p-PDA1. The URA3 is disrupted if desired by plating on 5-fluorootic acid (5FOA; Zymo Research, Orange, Calif.) using standard yeast techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Example 12 (Prophetic) Production of Isobutanol in Recombinant S. Cerevisiae [BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 GAL1p-PDA1]

The purpose of this prophetic example is to describe how to obtain isobutanol production in a yeast strain that is disrupted for pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase activities, has suppression of pyruvate dehydrogenase activity through use of the galactose promoter, and expresses cytosolic acetolactate synthase.

Plasmid vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5+GPMp-kivD (see Example 6) are transformed into strain BY4741 pdc1::FBAp-alsS-LEU2 pdc5::kanMX4 Δgpd1 Δgpd2 GAL1p-PDA1 (Example 11) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).and maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol. Aerobic cultures are grown in 250 ml flasks containing 50 ml synthetic complete media (minus histidine and uracil) supplemented with 2% glucose and 0.5% ethanol in an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Low oxygen cultures are prepared by adding 45 mL of medium to 60 mL serum vials that are sealed with crimped caps after inoculation and kept at 30° C. Approximately 24 h and 48 h after induction with 0.03 mM CuSO₄ (final concentration), an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection) and GC(HP-Innowax, 0.25 mm×0.2 μm×25 m (Agilent Technologies, Inc., Santa Clara, Calif.) with flame ionization detection (FID) for isobutanol content. Isobutanol is detected.

Example 13 Construction of Expression Vectors for Isobutanol Pathway Gene Expression in S. Cerevisiae pLH475-Z4B8 Construction

The pLH475-Z4B8 plasmid (SEQ ID NO:165) was constructed for expression of ALS and KARI in yeast. pLH475-Z4B8 is a pHR81 vector (ATCC #87541) containing the following chimeric genes:

-   1) the CUP1 promoter (SEQ ID NO: 125), acetolactate synthase coding     region from Bacillus subtilis (AlsS; SEQ ID NO:3; protein SEQ ID     NO:4) and CYC1 terminator (CYC1-2; SEQ ID NO:166); -   2) an ILV5 promoter (SEQ ID NO:167), Pf5.IlvC-Z4B8 coding region     (SEQ ID NO:168; protein SEQ ID NO:169) and ILV5 terminator (SEQ ID     NO:170); and -   3) the FBA1 promoter (SEQ ID NO: 116), S. cerevisiae KARI coding     region (ILV5; SEQ ID NO: 37; protein SEQ ID NO:38) and CYC1     terminator.

The Pf5.IlvC-Z4B8 coding region is a sequence encoding KARI derived from Pseudomonas fluorescens but containing mutations, that was described in commonly owned and co-pending U.S. patent application Ser. No. 12/337,736, which is herein incorporated by reference. The Pf5.1IlvC-Z4B8 encoded KARI (SEQ ID NO:169) has the following amino acid changes as compared to the natural Pseudomonas fluorescens KAR1:

-   C33L: cysteine at position 33 changed to leucine, -   R47Y: arginine at position 47 changed to tyrosine, -   S50A: serine at position 50 changed to alanine, -   T52D: threonine at position 52 changed to asparagine, -   V53A: valine at position 53 changed to alanine, -   L61F: leucine at position 61 changed to phenylalanine, -   T80I: threonine at position 80 changed to isoleucine, -   A156V: alanine at position 156 changed to threonine, and -   G170A: glycine at position 170 changed to alanine.

The Pf5.IlvC-Z4B8 coding region was synthesized by DNA 2.0 (Palo Alto, Calif.; SEQ ID NO:6) based on codons that were optimized for expression in Saccharomyces cerevisiae.

Expression Vector pLH468

The pLH468 plasmid (SEQ ID NO:171) was constructed for expression of DHAD, KivD and HADH in yeast.

Coding regions for B. subtilis ketoisovalerate decarboxylase (KivD) and Horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0 based on codons that were optimized for expression in Saccharomyces cerevisiae (SEQ ID NO:172 and 174, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs 173 and 175, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426::P_(GPD1)-kivDy-GPD1t), vector pNY8 (SEQ ID NO:176; also named pRS426.GPD-ald-GPDt, described in commonly owned and co-pending US Patent App. Pub. US2008/0182308, Example 17, which is herein incorporated by reference) was digested with AscI and SfiI enzymes, thus excising the GPD1 promoter (SEQ ID NO:114) and the ald coding region. A GPD1 promoter fragment (GPD1-2; SEQ ID NO:177) from pNY8 was PCR amplified to add an AscI site at the 5′ end, and an SpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067 (SEQ ID NOs:178 and 179). The AscI/SfiI digested pNY8 vector fragment was ligated with the GPD1 promoter PCR product digested with AscI and SpeI, and the SpeI-SfiI fragment containing the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. The triple ligation generated vector pLH467 (pRS426::P_(GPD1)-kivDy-GPD1t). pLH467 was verified by restriction mapping and sequencing.

pLH435 (pRS425::P_(GPM1)-Hadhy-ADH1t) was derived from vector pRS425::GPM-sadB (SEQ ID NO:180) which is described in commonly owned and co-pending U.S. Patent App. No. 61/058,970, Example 3, which is herein incorporated by reference. pRS425::GPM-sadB is the pRS425 vector (ATCC #77106) with a chimeric gene containing the GPM1 promoter (SEQ ID NO:135), coding region from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB; SEQ ID NO: 35; protein SEQ ID NO:36: disclosed in commonly owned and co-pending U.S. Patent App. No. 61/048,291), and ADH1 terminator (SEQ ID NO:126). pRS425::GPMp-sadB contains BbvI and PacI sites at the 5′ and 3′ ends of the sadB coding region, respectively. A NheI site was added at the 5′ end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NO:181 and 182) to generate vector pRS425-GPMp-sadB-NheI, which was verified by sequencing. pRS425::P_(GPM1)-sadB-NheI was digested with NheI and PacI to drop out the sadB coding region, and ligated with the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadhy-DNA2.0 to create pLH435.

To combine KivD and HADH expression cassettes in a single vector, yeast vector pRS411 (ATCC #87474) was digested with SacI and NotI, and ligated with the SacI-SalI fragment from pLH467 that contains the P_(GPD1)-kivDy-GPD1t cassette together with the SalI-NotI fragment from pLH435 that contains the P_(GPM1)-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the vector pRS411::P_(GPD1)-kivDy-P_(GPM1)-Hadhy (pLH441), which was verified by restriction mapping.

In order to generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy and Hadhy, we used pRS423 FBA ilvD(Strep) (SEQ ID NO:183), which is described in commonly owned and co-pending U.S. Patent Application No. 61/100,792, as the source of the IlvD gene. This shuttle vector contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector has an FBA promoter (nt 2111 to 3108; SEQ ID NO:10) and FBA terminator (nt 4861 to 5860; SEQ ID NO:184). In addition, it carries the His marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO:185; protein SEQ ID NO:186) from Streptococcus mutans UA159 (ATCC #700610) is between the FBA promoter and FBA terminator forming a chimeric gene for expression. In addition there is a lumio tag fused to the ilvD coding region (nt 4829-4849).

The first step was to linearize pRS423 FBA ilvD(Strep) (also called pRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio) with SacI and SacII (with SacII site blunt ended using T4 DNA polymerase), to give a vector with total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI site blunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment. This fragment was ligated with the 9,482 bp vector fragment from pRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio. This generated vector pLH468 (pRS423::P_(FBA1)-ilvD(Strep)Lumio-FBA 1t-P_(GPD1)-kivDy-GPD1t-P_(GPM1)-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.

Example 14 Pyruvate Decarboxylase Gene Inactivation

This example describes insertion-inactivation of endogenous PDC1, PDC5, and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase. The resulting PDC inactivation strain was used as a host for expression vectors pLH475-Z4B8 and pLH468 that were described in Example 13.

Construction of pdc6::GPMp1-sadB Integration Cassette and PDC6 Deletion:

A pdc6::GPM1p-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO:187) from pRS425::GPM-sadB (described above) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:188) contains the URA3 marker from pRS426 (ATCC #77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 114117-11A through 114117-11D (SEQ ID NOs:189, 190, 191 and 192), and 114117-13A and 114117-13B (SEQ ID NOs:193 and 194).

The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′ ˜50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC #200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs:195 and 196), and 112590-34F and 112590-49E (SEQ ID NOs: 113 and 197) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::P_(GPM1)-sadB-ADH1t.

Construction of pdc1::PDC1-ilvD Integration Cassette and PDC1 Deletion:

A pdc1::PDC1p-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO:198) from pLH468 (described above) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 114117-27A through 114117-27D (SEQ ID NOs:199, 200, 201 and 202).

The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′ ˜50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::P_(GPM1)-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs 203 and 204), and primers 112590-49E and 112590-30F (SEQ ID NOs 197 and 205) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t.

HIS3 Deletion

To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO;206). URA3r2 contains the URA3 marker from pRS426 (ATCC #77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion DNA polymerase and primers 114117-45A and 114117-45B (SEQ ID NOs:207 and 208) which generated a ˜2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA73” has the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3.

Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs:106 and 107) which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 108 and 109). The identified correct transformants have the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3 pdc5::kanMX4.

Plasmid vectors pLH468 and pLH475-Z4B8 were simultaneously transformed into strain BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3 pdc5::kanMX4 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the resulting strain was maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol at 30° C. The resulting strain was named NGI-049.

Example 15 Production of Isobutanol by Saccharomyces cerevisiae Strain NGI-049

A seed culture of NGI-049 for inoculum preparation was grown in Yeast Nitrogen Base (YNB) without amino acids medium (6.7 g/L), supplemented with amino acid dropout mix (1.4 g/L), leucine (100 mg/L) and tryptophan (20 mg/L). Ethanol at 1% (v/v) was used as the sole carbon source for seed cultures. The fermentation medium was a semi-synthetic medium, the composition of which is given in Table 7.

TABLE 7 Fermentation Medium Composition Ingredient Amount/L 1. YNB w/o amino acids ^(a) 6.7 g 2. Sigma Dropout Mix (Y2001) ^(b) 2.8 g 3. Leucine (10 g/L) 20 ml 4. Tryptophan (10 g/L) 4 ml 5. Ethanol 10 ml 6. Glucose 50 wt % stock 4 g ^(a) Obtained from BD Diagnostic Systems, Sparks, MD ^(b) Obtained from Sigma-Aldrich, St. Louis, MO Ingredients 1-4 from Table 7 were added to water at the prescribed concentration to make a final volume of 0.54 L in the fermentor. The contents of the fermentor were sterilized by autoclaving. Components 5 and 6 were mixed, filter sterilized and added to the fermentor after the autoclaved medium had cooled. The total final volume of the fermentation medium (the aqueous phase) was about 0.54 L.

The fermentation was done using a 1 L autoclavable bioreactor, Bio Console ADI 1025 (Applikon, Inc, Holland) with a working volume of 900 mL. The temperature was maintained at 30° C. during the entire fermentation and the pH was maintained at 5.5 using sodium hydroxide. Following inoculation of the sterile fermentation medium with seed culture (10 vol %), the fermentor was operated aerobically at a 30% dissolved oxygen (DO) set point with 0.3 vvm of air flow by automatic control of the agitation rate (rpm). Once the initial batched glucose of 2 g/L was consumed, glucose was fed using a pump at an exponential rate such that glucose never accumulated above 0.2 g/L in the fermentor. Once the desired optical density (OD₆₀₀) was reached (i.e., OD₆₀₀=6), the culture was induced to isobutanol production phase by feeding glucose such that excess glucose (>2 g/L) was maintained at all times during fermentation. Two hours post glucose excess, 60 mL of filter sterilized 10× Yeast Extract Peptone stock solution (10×YEP=100 g/L of yeast extract and 200 g/L of peptone) was added. Glucose was fed (50 wt % stock solution) to the fermentor to keep levels of glucose greater than 2 g/L.

Because efficient production of isobutanol requires microaerobic conditions to enable redox balance in the biosynthetic pathway, air was continuously supplied to the fermentor at 0.3 vvm. Continuous aeration led to significant stripping of isobutanol from the aqueous phase of the fermentor. To quantify the loss of isobutanol due to stripping, the off-gas from the fermentor was directly sent to a mass spectrometer (Prima dB mass spectrometer, Thermo Electron Corp., Madison, Wis.) to quantify the amount of isobutanol in the gas stream. The isobutanol peaks at mass to charge ratios of 74 or 42 were monitored continuously to quantify the amount of isobutanol in the gas stream.

Glucose and organic acids in the aqueous phase were monitored during the fermentation using HPLC. Glucose was also monitored quickly using a glucose analyzer (YSI, Inc., Yellow Springs, Ohio). Isobutanol in the aqueous phase was quantified by HPLC as described in the General Methods Section herein above after the aqueous phase was removed periodically from the fermentor. For isobutanol production, the effective titer, the effective rate, and the effective yield, all corrected for the isobutanol lost due to stripping, were 3 g/L, 0.04 g/L/h, and 0.16 g/g, respectively. 

What is claimed:
 1. A recombinant microorganism for producing isobutanol, the recombinant yeast microorganism comprising an engineered isobutanol biosynthetic pathway, wherein said engineered isobutanol biosynthetic pathway comprises the following substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate 2,3-dihydroxy-isovalerate; (c) 2,3-dihydroxy-isovalerate to α-ketoisovalerate; and (d) α-ketoisovalerate to isobutyraldehyde; wherein i) the substrate to product conversion of step (a) is performed by an acetolactate synthase enzyme; ii) the substrate to product conversion of step (b) is performed by an acetohydroxy acid isomeroreductase enzyme; iii) the substrate to product conversion of step (c) is performed by an acetohydroxy acid dehydratase enzyme; and iv) the substrate to product conversion of step (d) is performed by a branched-chain α-keto decarboxylase; and wherein the recombinant yeast microorganism is substantially free of at least one enzyme having pyruvate decarboxylase activity and is substantially free of at least one enzyme having glycerol-3-phosphate dehydrogenase activity.
 2. The recombinant yeast of claim 1, wherein the engineered isobutanol biosynthetic pathway further comprises the following substrate to product conversion: (e) isobutyraldehyde to isobutanol wherein the substrate to product conversion of step (e) is performed by an alcohol dehydrogenase enzyme.
 3. The recombinant yeast of claim 1, wherein the conversion of pyruvate to acetolactate is catalyzed by a cytosol-localized polypeptide having acetolactate synthase activity.
 4. The recombinant yeast of claim 3, wherein the polypeptide having acetolactate synthase activity has a substrate preference for pyruvate over ketobutyrate.
 5. The recombinant yeast of claim 3, wherein the polypeptide having acetolactate synthase activity is a Lactococcas lactis acetolactate synthase.
 6. The recombinant yeast of claim 3, wherein the polypeptide having acetolactate synthase activity is a Bacillus subtilis acetolactate synthase.
 7. The recombinant yeast of claim 1, wherein the yeast cell comprises a disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or controlling pyruvate decarboxylase gene expression.
 8. The recombinant yeast of claim 7, wherein the polypeptide is selected from the group consisting of PDC1, PDC2, PDC5, and PDC6.
 9. The recombinant yeast of claim 8, comprising a disruption in PDC1 and PDC5.
 10. The recombinant yeast of claim 1, comprising a disruption in at least one gene encoding an NAD-dependent glycerol-3-phosphate dehydrogenase selected from the group consisting of GPD1 and GPD2.
 11. The recombinant yeast of claim 1, wherein the conversion of pyruvate to acetolactate is at least about 60% of theoretical yield.
 12. The recombinant yeast of claim 1, further comprising a balance in reducing equivalents, wherein the conversion of pyruvate to acetolactate is at least about 86% of theoretical yield.
 13. The recombinant yeast of claim 1 wherein the yeast further comprises a construct for heterologous expression of a secondary alcohol dehydrogenase.
 14. The recombinant yeast of claim 1, wherein the yeast is Kluyveromyces lactis.
 15. The recombinant yeast of claim 1, wherein the yeast is Saccharomyces cerevisiae.
 16. A method of producing isobutanol, comprising: (a) providing, the recombinant yeast microorganism according to claim 1; and (b) growing the recombinant yeast microorganism under conditions where isobutanol is produced.
 17. The method of claim 16 further comprising (c) recovering the isobutanol. 